A variety of enzyme immunoassay techniques are known. These include methods wherein an enzyme is bound to an antibody or antigen to be detected. Competition between the enzyme labeled species and unknown for the binding partner bound to a solid support is measured. The enzyme remaining in the solution is measured by reaction to substrate.
Homogeneous enzyme immunoassay techniques are described in U.S. Pat. No. 4,043,872 (hapten bound to enzyme) and U.S. Pat. No. 4,065,354 (hapten bound to lysoyme). Enzyme cofactor labeled ligand are described in Analytical Biochemistry 72, 271 and 283 (1976). Derwendt Abstract B4, Natural Products Week A26, pp. 30 of DT2754-086 describes reversible binding enzyme modulators as a labeling substance for antigen, antibody, hormone, vitamins or drugs. Belgium Pat. No. 864,856, Sept. 9, 1978, describes conjugates using methotrexate as an enzyme inhibitor bound to ligand analogs.
Methods and reagents of the present invention are particularly distinct in that they involve the use of irreversible enzyme inhibitors conjugated to ligand analogs. The inhibitors of the present invention react with the enzymes forming covalent bonds altering the structure of the enzyme and thereby irreversibly inhibiting the enzyme activity. In particular, organophosphorous irreversible enzyme inhibitors which react with an enzyme to form covalent bonds are conjugated to ligand analogs.